Various naturally occurring aromatic carboxylic acids have been found to have utility in industrial applications. Compounds such as para-hydroxycinnamic acid (PHCA) and para-hydroxybenzoic acid (PHBA) are high-value, compounds that may be used as monomers for the production of Liquid Crystal Polymers (LCP). LCPs are polymers that exhibit an intermediate or mesophase between the glass-transition temperature and the transition temperature to the isotropic liquid or have at least one mesophase for certain ranges of concentration and temperature. The molecules in these mesophases behave like liquids and flow, but also exhibit the anisotropic properties of crystals. LCPs are used in liquid crystal displays, and in high speed connectors and flexible circuits for electronic, telecommunication, and aerospace applications. Because of their resistance to sterilizing radiation and their high oxygen and water vapor barrier properties, LCPs are used in medical devices, and in chemical and food packaging.
Methods for the chemical synthesis of PHCA and PHBA are known. However, chemical synthesis is expensive due to the high energy needed for synthesis and the extensive product purification required. Biological production of these compounds offers a low cost, simplified solution to the problem.
Several methods of producing aromatic carboxylic acids from recombinant microorganisms are described in the literature (see for example commonly owned U.S. Ser. No. 10/439,479; U.S. Pat. No. 6,368,837; and U.S. Pat. No. 6,521,748). However it will be advantageous to optimize production of these molecules for commercial use. One route to optimized production is increased yield. As many of these aromatic carboxylic acids are toxic to the producing host cell, another route may be to minimize the toxic effect the end product has on the host cell. A family of ubiquitous proteins that may be able to address both of these issues are the efflux proteins.
Cellular production of biomolecules can be optimized, in part, by optimizing the expression of efflux transport proteins in the production strain. For example, increased expression of efflux systems for toxic products may be critical for achieving desired rate, titer and yield.
Over 200 transport protein families have been identified, with more than 100 of these transport protein families existing in bacteria (Saier et al., FASEB J. 12:265-274 (1998)). At least four superfamilies of drug resistance transporters are known to exist in bacteria. These superfamilies are the ATP Binding Cassette superfamily, the Major Facilitator Superfamily, the Drug/Metabolite Transporter superfamily (Jack et al., Eur. J. Biochem. 268:3620-3639 (2001)), and the Resistance-Nodulation-Cell Division family.
Overexpression of an efflux system or its expression from a plasmid vector results in increased resistance of bacteria to a variety of toxic substances, while inactivation of an efflux system causes an increase in sensitivity to antibiotics and toxic substances (Li et al., J. Bacteriol. 180:2987-2991(1998); Ramos, et al., J. Bacteriol. 180:3323-3329 (1998)). Such efflux systems are increasingly being recognized in a wide range of bacteria. Comparative amino acid sequence analysis of various transport proteins plus function assays has enabled the identification of a number of distinct families and superfamilies of transport proteins.
U.S. Pat. Nos. 6,225,089 and 6,235,882 issued to Chen (May, 2001) disclose the isolation of a gene encoding a putative efflux protein for solvents and antibiotics. The putative efflux protein, isolated from Pseudomonas mendocina (“P. mendocina”), is used to examine efflux systems related to solvent tolerance. Culturing P. mendocina strains containing altered levels of the gene encoding the putative efflux protein in medium containing increased levels of para-hydroxybenzoic acid results in accumulation of para-hydroxybenzoic acid. The putative efflux protein contains highly conserved regions or motifs that are indicative of proteins in the Major Facilitator Superfamily.
Tolerance of bacteria cells containing efflux pump mutants to para-hydroxybenzoic acid is indicative of the involvement of these efflux pumps in para-hydroxybenzoic acid extrusion (Godoy et al., J. Bacteriol. 183:5285-5292 (2001); Ramos-Gonzalez et al., Appl. Environ. Microbiol. 67:4338-4341 (2001)). In Pseudomonas putida (“P. putida”), two efflux pumps, TtgABC and TtgDEF, are speculated to be involved in extrusion of para-hydroxybenzoic acid. Mutation of these efflux pumps, in coordination with increased rigidity of the cell membrane, results in the accumulation of para-hydroxybenzoic acid in P. putida strains.
PcaK, a protein also isolated from P. putida, is a transporter responsible for the influx of para-hydroxybenzoic acid (Ditty and Harwood, J. Bacteriol. 181:5068-5074 (1999)). This transporter, a member of the Major Facilitator Superfamily, also participates in chemotaxis to extracellular para-hydroxybenzoic acid. PcaK does not, however, transport benzoic acid into the cell. Expression of wild-type PcaK protein in Escherichia coli (“E. coli”) results in increased accumulation of para-hydroxybenzoic acid compared to E. coli expressing a PcaK mutant.
U.S. Pat. No. 5,292,643 issued to Shibano et al. on Mar. 8, 1994 describes genes related to fusaric acid resistance in a variety of microorganisms. Specifically, genes capable of decomposing or detoxifying fusaric acid are disclosed. One of the genes postulated to be involved in fusaric acid resistance, fusB, shares some homology with the PET yhcP gene (Paulsen et al., FEMS Microbiol. Lett. 156:1-8 (1997)).
Applicants incorporate by reference the co-owned and concurrently filed application entitled “Regulator/Promoter for Tunable Gene Expression and Metabolite Sensing”, U.S. Patent Application No. 60/440,965.
Recently, the PET family of proteins in bacteria, yeast, and green plants was identified using bioinformatics techniques (Harley and Saier, J. Mol. Microbiol. Biotechnol. 2:195-198 (2000)).
The problem to be solved therefore is to enhance the production of aromatic carboxylic acids without compromising the production host due to increased toxicity to the end product. Applicants have solved the stated problem through the discovery that a family of efflux proteins encoded by the yhcRQP operon, both increases the flux of the carboxylic acids from the cell to the medium and lowers toxicity of the cell to the carboxylic acid end product.